Truncations of the cytoplasmic tail of the HIV-1 transmembrane (TM) protein are rare and almost always markedly reduce virus infectivity. We describe a truncation of the gp41 cytoplasmic tail in the commonly used early HIV-1 reference strain RF. This truncation apparently arose after continuous passage in H9 cells. We detected the truncation by Western blot as a size decrease in RF gp41 from 46 to approximately 34 kDa. The reduced size of RF gp41 observed was not due to differences in glycosylation. Viral DNA sequencing confirmed that a point mutation at Env residue 740 (Trp) introduced a premature stop codon, resulting in a 100-amino acid (13-kDa) truncation of the gp41 C terminus. This truncated RF species, termed RF(gp34), was characterized phenotypically by growth in Hut78 cells. Compared with other B clade HIV strains (IIIB, SF2, and NL4.3), RF(gp34) induced massive syncytia. Importantly, RF(gp34) also productively infected peripheral blood mononuclear cells in vitro.